Association free energy of dipalmitoylphosphatidylserines in a mixed dipalmitoylphosphatidylcholine membrane Blood coagulation is strongly dependent on the binding of vitamin K-dependent proteins to cell membranes containing phosphatidylserine (PS) via g-carboxyglutamic acid (Gla) domains. The process depends on calcium, which can induce non-ideal behavior in membranes through domain formation. Such domain separation mediated by Ca²⁺ ions or proteins can have an important contribution to the thermodynamics of the interaction between charged peripheral proteins and oppositely charged membranes. To characterize the properties of lipid-lipid interactions, molecular dynamics and free energy simulations in a mixed bilayer membrane containing DPPC and DPPS were carried out. The free energy of association between DPPSs in the environment of DPPCs has been calculated by using a novel approach to the dual topology technique of the PS-PC hybrid. Two different methods, free energy perturbation (FEP) and thermodynamic integration (TI), were used to calculate the free energy difference. In TI runs three schemes were applied to evaluate the integral at the limits of l -> 0 or l -> 1. Our studies show that the association of two PSs in the environment of PCs is repulsive in the absence of Ca²⁺ and becomes favorable in their presence. We also show that the mixed component membrane should exhibit non-ideal behavior that will lead to PS clustering.